Probes have been developed for a variety of diagnostic and research purposes. Hybridization of chromosome or gene-specific probes has made possible detection of chromosomal abnormalities associated with numerous diseases and syndromes, including constitutive genetic anomalies (such as microdeletion syndromes, chromosome translocations, gene amplification and aneuploidy syndromes), neoplastic diseases, as well as pathogen infections. Detection of genetic changes in these regions can provide diagnostic and prognostic information for patients and in some cases, inform treatment decisions.
Dual detection and enumeration of human chromosome 17 (CHR17) and human epidermal growth factor receptor 2 (HER2) is important for the selection of appropriate patients for HER2 targeted therapy in breast cancer (Wolff A C, et al., J Clin Oncol 2007, 25:118-145; Gruver A M, et al., J Clin Pathol 2010 March; 63(3):210-9), but existing probes that may be used for such dual detection and enumeration are known for requiring long assay times to obtain specific and sensitive detection.
Double-stranded CHR17 centromere probes are typically generated from the p17H8 plasmid sequence, which is directed to human CHR17's alpha satellite. The alpha satellite of human CHR17 contains a ˜2,700 base pair higher order repeat unit that consists of 16 monomers and is present in 500 to 1,000 copies per CHR17 (Waye J S, et al., Molecular and Cellular Biology, September 1986, p. 3156-3165). Double-stranded HER2 probes are typically generated from bacterial artificial chromosomes (BACs) and span the HER2 gene (Dal Lago L, et al., Mol Cancer Ther 2006, 52572-2579; Gruver A M, et al., J Clin Pathol 2010 March; 63(3):210-9). These double-stranded probes have repetitive sequences that are common to centromere regions of other human chromosomes. Consequently, a significant drawback to these probes is the noise-generating repetitive elements. That is, probes to the centromere regions typically have significant cross-reactivity to other chromosome centromeres. As such, blocking DNA has been required to be used in conjunction with these probes to reduce non-specific binding (See Pinkel and Gray, U.S. Pat. No. 5,447,841). Assays employing these probes require extensive hybridization time to achieve sufficient hybridization because of their double-stranded nature and the required competition with the blocking DNA, e.g., about 6 to 18 hours. This time consuming step reflects low hybridization efficiency, in part due to self-hybridization of the double-stranded probe and in part because of the competition with the blocking DNA. Libraries of BAC probes are also cumbersome to generate and maintain, laborious to purify, and are prone to contamination. The benchmark and ground-breaking assay using this technology was disclosed by Nitta et al. in 2008 and is commercially available as the INFORM HER2 Dual ISH DNA Probe Cocktail, Ventana Medical Systems, Catalog Number: 780-4422 (Nitta et al. Diagnostic Pathology, 3:41, 2008).
Recently, Matthiesen and Hansen (Matthiesen S H, et al., PLoS One, 2012; 7(7), 2012) claimed that with no change in the HER2 and CHR17 probe configuration, substitution of ethylene carbonate (EC) for formamide in the hybridization buffer reduced FISH hybridization time and requires no blocking DNA. The HER2 IQFISH pharmDx™ assay (Dako) was introduced to the market based on this technology. While a useful technique, fluorescence in situ hybridization (FISH) has its drawbacks. Implementation of conventional FISH requires a dedicated fluorescence imaging system and well-trained personnel with specific expertise, making this system incompatible with some clinical workflows. Furthermore, when compared to bright-field in situ hybridization (ISH) approaches, FISH studies provide relatively limited morphological assessment of overall histology, lack stability of the fluorescent detection signal(s) over time, and have a higher overall cost of testing.
In an effort to alleviate drawbacks associated with clone-based probes, investigators have proposed the use of “specific primers” to generate probes from genomic DNA (Navin et al., Bioinformatics 222437-2438 (2006)). However, this process is cumbersome and time consuming in that it requires multiple specific amplification reactions and downstream processing with upfront hands-on time (See also Yamada et al., Cytogenet Genome Res. 1-7 (2010)).
For some applications, the use of single-stranded probes has a distinct advantage over the use of double-stranded probes. For example, single-stranded probes generally have higher sensitivity than double-stranded probes because a proportion of the denatured double-stranded probe renatures to form probe homoduplexes, thus preventing their capture of genomic targets in the test samples (Taneja K et al., Anal Biochem, 166, 389-398 (1987), Lewis M E, et al., Peptides, 6 Suppl 2:75-87 (1985); Strachan T, Read A P, Human Molecular Genetics. 2nd edition. New York: Wiley-Liss (1999); Kourilsky P, et al., Biochimie, 56(9):1215-21 (1974)). Several laboratories have reported that single-stranded probes provide higher hybridization sensitivity than double-stranded probes (An S F, et al., Mol Cell Probes, 6(3):193-200 (1992); Hannon K, et al, Anal Biochem, 212(2):421-7 (1993); Cox K H, et al., Dev Biol., 101(2):485-502 (1984)).
Synthetic single-stranded oligonucleotide probes have been used to detect genomic targets, mostly for FISH. For example, Bergstrom et al, Designing Custom Oligo FISH Probes for the Detection of Chromosomal Rearrangements in FFPE Tissues, American Society of Human Genetics 2013 Meeting (2013) reported SureFISH probes comprising thousands of unique, long single-stranded oligonucleotides with fluorescence labels. The oligonucleotide sequences tile across the targeted chromosomal region of translocation breakpoints for the detection of chromosomal rearrangements. Although Bergstrom discloses single-stranded probes, the probes were not directed to CHR17 and the Bergstrom reference does not appear to provide any solutions to the difficulties associated with CHR17 probes, such as specificity and robustness to detect CHR17 polymorphisms in a human population. Also, the Bergstrom reference does not disclose assays (and probes) for gene copy number enumeration wherein a target probe and a reference probe are used in combination to calculate a target gene to reference chromosome ratio.
The use of single-stranded oligonucleotide probes for genomic targets has been extremely limited. For example, U.S. Pat. No. 8,445,206 (Bergmann et al., 2012) describes a set of at least 100 single-stranded oligonucleotide probes directed against (or complementary to) portions of the HER2 gene. The disclosure appears to be limited to detection of the HER2 gene target without a reference probe (e.g., CHR17), which is useful for gene copy number assessment as the HER2/CHR17 ratio is diagnostically important as evidenced from the teachings of Wolff A C, et al., J Clin Oncol 2007, 25:118-145.
Comparative genome hybridization (CGH) assays may be used for providing information on the relative copy number of one sample (such as a tumor sample) compared to another (such as a reference sample, for example a non-tumor cell or tissue sample). Thus, CGH may be used for determining whether genomic DNA copy number of a target nucleic acid is increased or decreased as compared to the reference sample. However, CGH does not provide information as to the exact number of copies of a particular genomic DNA or chromosomal region.
For genomic labeling of CHR17, a previous 42-mer oligonucleotide derived from p17H8 was demonstrated to be specific to CHR17. But, because of significant differences in the sizes of the 42-mer CHR17 probe and the preferred oligomeric HER2 probes (ranging from about 100 bp to about 400 bp) disclosed herein, the dual HER2-CHR17 ISH assay required a lengthy procedure to sequentially detect HER2 and CHR17 signals under different stringency wash temperatures (72° C. for HER2 and 59° C. for CHR17). Importantly, dual ISH experiments using the 42-mer CHR17 probe and single-stranded HER2 probes of a similar size did not resolve the incompatibility of the probe sets (See FIG. 14A-D and Example 2). Further, even if the incompatibility between the 42-mer CHR17 probe and the single stranded HER2 probes were resolved, a single oligonucleotide probe (e.g., the 42-mer CHR17 probe) specific for only a one monomer of the alpha satellite's 16 monomers as taught by Nitta would not be sufficient to detect CHR17 throughout the human population since each individual human being may carry different combinations of the monomers and their related variants (Waye J S and Willard H F, NAC 1986; 14(17); Willard, H. F. et al, 1987, Genomics, 1; Warburton, P. E. and Willard, H. F., 1995, J. Mol. Evol., 41). Thus, the 42-mer CHR17 probe as taught by Nitta would not be robust enough across the entire population.
Despite the appeal of the use of a single-stranded CHR17 probe, workers in this field thought it is not possible to make short, single-stranded CHR17 probes that are specific enough to CHR17 (e.g., specific enough to eliminate the need for blocking DNA), and robust enough to sufficiently detect CHR17 throughout the human population. One of the reasons for this understanding is that it was believed that the fundamental repetitive nature of alpha satellite DNA makes the likelihood of finding short oligonucleotides specific enough to CHR17 impossibly improbable. For example, Willard (Willard, H. F., 1985, Am J Hum Genet, 37; Willard, H. F., 1991, Curr Opin Genet Dev. 1) found sequences of the same monomer in different higher order repeat units that showed a level of similarity approaching 99%. Further, there appear to be a significant number of off-target hits to other chromosomes. For example, bioinformatics research revealed that 14 oligonucleotide sequences derived from plasmid p17H8 (comprising the higher order repeat units in the centromere region of CHR17) had high homology to several other chromosomes (e.g., chromosome 1, X, 11, 9, 20, 22, etc.). Although a number of sequences of each oligonucleotide had high homology (85-100%) to CHR17, there were also many off-target hits. For instance, a representative oligonucleotide (M2.1) had 21 on-target hits but also had 33 hits on chromosome 1; another oligonucleotide (M2.2) had 18 on-target hits but also had 14 hits on chromosome X (See FIG. 15). These results suggest that the centromere region of CHR17 may not contain sufficiently specific sequences for targeting. Indeed, examining the centromere region from a bioinformatics perspective indicates that designing probes uniquely specific to the centromere, which would be capable of providing selective signal without the use of blocking DNA, is not reasonable or expected to be possible.
Another reason that workers in the field expected it was not possible to make short, single-stranded CHR17 probes specific enough to CHR17 (e.g., specific enough to eliminate the need for blocking DNA) is because of the lack of robustness of a single (or a few number of) single-stranded oligonucleotide probe(s). As discussed above, human CHR17-specific alpha satellite contains a higher order repeat unit that consists of 16 monomers, and each individual human being may carry different combinations of these monomers and their related variants (Waye J S and Willard H F, Molecular and Cellular Biology, September 1986, p. 3156-3165). A single oligonucleotide probe, e.g. the 42mer described above, or even a few number of oligonucleotides covering a small number of monomers, may not be robust enough to detect CHR17 polymorphism in a human population (Waye J S, Willard H F., NAC 1986; 14(17); Willard, H. F. et al, 1987, Genomics, 1; Warburton, P. E. and Willard, H. F., 1995, J. Mol. Evol., 41). Indeed, a single CHR17-specific oligonucleotide probe (79mer) did not show equivalent (or better) sensitivity to the p17H8 plasmid derived probe. In particular, when the single 79mer CHR17 oligonucleotide was compared to the commercial probe (p17H8 probe), it was found that it passed (signal intensity ≥2, coverage ≥50%, and background<2) only 41.5% (113/272) at 1 μg/mL, 1 hr compared to 61.1% (148/242) at 0.75 μg/mL, 6 hr. Accordingly, the Chr17 Oligonucleotide (a single 79mer) failed to show equivalent sensitivity to the commercial probe design.
Another reason that workers in the field expected it was not possible to make short, single-stranded CHR17 probes specific enough for CHR17 was because the making of such oligonucleotide probes is very cumbersome and the manufacturability of such product is heretofore, not readily known. In particular, to span a 1 million bp genomic region with probes hybridizing to at least 60 kb of target, as many as 1200 unique 50-mer oligonucleotide probes may be needed. Manufacturing 1200 unique probes and combining them within a single reagent is difficult, expensive, and breaks new ground from a regulatory perspective.